LOCUS       sequence-194720-        5546 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5546)
  TITLE     SGC Toronto Q2 Q3 2016
REFERENCE   2  (bases 1 to 5546)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: 5T1G (CBX1)
FEATURES             Location/Qualifiers
     source          1..5546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             91..108
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             121..141
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             411..428
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(481..499)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      495..542
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      579..1034
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(666..685)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     876..897
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1126..1941)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1126..1941)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1849..1868
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1933..1952
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2063..2651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2552..2571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2805..2822
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2837..2976
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2902..2921)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3062..3084
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3078..3269)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4078..5160)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5122..5141
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5161..5238)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5444..5463
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg catcatcatc atcatcacag cagcggcaga
      121 gaaaacttgt atttccaggg cgagtcagaa aagccacgag gctttgctcg aggtttggag
      181 ccggagcgga ttattggagc tacagactcc agtggagagc tcatgttcct gatgaaatgg
      241 aaaaactctg atgaggctga cctggtccct gccaaggaag ccaatgtcaa gtgcccacag
      301 gttgtcatat ccttctatga ggaaaggctg acgtggcatt cctacccctc ggaggatgat
      361 gacaaaaaag atgacaagaa ctgatgacga agcttgcggc cgcactcgag caccaccacc
      421 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
      481 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
      541 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
      601 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
      661 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      721 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      781 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
      841 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
      901 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
      961 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1021 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1081 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     1141 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     1201 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     1261 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     1321 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     1381 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1441 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1501 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1561 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1621 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     1681 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     1741 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     1801 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     1861 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     1921 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     1981 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2041 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2101 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2161 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2221 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2281 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2341 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2401 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2461 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2521 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2581 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2641 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2701 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2761 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2821 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2881 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     2941 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3001 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3061 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3121 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3181 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3241 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3301 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3361 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3421 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3481 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3541 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3601 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3661 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3721 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     3781 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     3841 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     3901 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     3961 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4021 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4081 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4141 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4201 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4261 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4321 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     4381 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4441 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4501 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4561 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4621 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4681 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4741 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4801 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     4861 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     4921 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     4981 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5041 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5101 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5161 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5221 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5281 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5341 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5401 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5461 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     5521 ggatcgagat ctcgatcccg cgaaat
//
